Proteins exhibit structural fluctuations over decades of time scales. From the picosecond side chain motions to aggregates that form over the course of minutes, characterizing protein structure over ...these vast lengths of time is important to understanding their function. In the past 15 years, two-dimensional infrared spectroscopy (2D IR) has been established as a versatile tool that can uniquely probe proteins structures on many time scales. In this review, we present some of the basic principles behind 2D IR and show how they have, and can, impact the field of protein biophysics. We highlight experiments in which 2D IR spectroscopy has provided structural and dynamical data that would be difficult to obtain with more standard structural biology techniques. We also highlight technological developments in 2D IR that continue to expand the scope of scientific problems that can be accessed in the biomedical sciences.
There is an enormous amount of interest in the structures and formation mechanisms of amyloid fibers. In this Perspective, we review the most common structural motifs of amyloid fibers and discuss ...how infrared spectroscopy and isotope labeling can be used to identify their structures and aggregation kinetics. We present three specific strategies, site-specific labeling to obtain residue-by-residue structural information, isotope dilution of uniformly labeled proteins for identifying structural folds and protein mixtures, and expressed protein ligation for studying the domain structures of large proteins. For each of these methods, vibrational couplings are the source of the identifying features in the infrared spectrum. Examples are provided using the proteins hIAPP, Aβ, polyglutamine, and γD-crystallin. We focus on FTIR spectroscopy but also describe new observables made possible by 2D IR spectroscopy.
Vibrational and electronic transition dipole strengths are often good probes of molecular structures, especially in excitonically coupled systems of chromophores. One cannot determine transition ...dipole strengths using linear spectroscopy unless the concentration is known, which in many cases it is not. In this paper, we report a simple method for measuring transition dipole moments from linear absorption and 2D IR spectra that does not require knowledge of concentrations. Our method is tested on several model compounds and applied to the amide I(') band of a polypeptide in its random coil and α-helical conformation as modulated by the solution temperature. It is often difficult to confidently assign polypeptide and protein secondary structures to random coil or α-helix by linear spectroscopy alone, because they absorb in the same frequency range. We find that the transition dipole strength of the random coil state is 0.12 ± 0.013 D(2), which is similar to a single peptide unit, indicating that the vibrational mode of random coil is localized on a single peptide unit. In an α-helix, the lower bound of transition dipole strength is 0.26 ± 0.03 D(2). When taking into account the angle of the amide I(') transition dipole vector with respect to the helix axis, our measurements indicate that the amide I(') vibrational mode is delocalized across a minimum of 3.5 residues in an α-helix. Thus, one can confidently assign secondary structure based on exciton delocalization through its effect on the transition dipole strength. Our method will be especially useful for kinetically evolving systems, systems with overlapping molecular conformations, and other situations in which concentrations are difficult to determine.
We have developed a broad bandwidth two-dimensional electronic spectrometer that operates shot-to-shot at repetition rates up to 100 kHz using an acousto-optic pulse shaper. It is called a ...two-dimensional white-light (2D-WL) spectrometer because the input is white-light supercontinuum. Methods for 100 kHz data collection are studied to understand how laser noise is incorporated into 2D spectra during measurement. At 100 kHz, shot-to-shot scanning of the delays and phases of the pulses in the pulse sequence produces a 2D spectrum 13-times faster and with the same signal-to-noise as using mechanical stages and a chopper. Comparing 100 to 1 kHz repetition rates, data acquisition time is decreased by a factor of 200, which is beyond the improvement expected by the repetition rates alone due to reduction in 1/f noise. These improvements arise because shot-to-shot readout and modulation of the pulse train at 100 kHz enables the electronic coherences to be measured faster than the decay in correlation between laser intensities. Using white light supercontinuum for the pump and probe pulses produces high signal-to-noise spectra on samples with optical densities <0.1 within a few minutes of averaging and an instrument response time of <46 fs thereby demonstrating that that simple broadband continuum sources, although weak, are sufficient to create high quality 2D spectra with >200 nm bandwidth.
Two-dimensional (2D) optical spectroscopy contains cross-peaks that are helpful features for determining molecular structure and monitoring energy transfer, but they can be difficult to resolve from ...the much more intense diagonal peaks. Transient absorption (TA) spectra contain transitions similar to cross-peaks in 2D spectroscopy, but in most cases they are obscured by the bleach and stimulated emission peaks. We report a polarization scheme, <0°,0°,+θ
(t
),-θ
(t
)>, that can be easily implemented in the pump-probe beam geometry, used most frequently in 2D and TA spectroscopy. This scheme removes the diagonal peaks in 2D spectroscopies and the intense bleach/stimulated emission peaks in TA spectroscopies, thereby resolving the cross-peak features. At zero pump-probe delay, θ
= 60° destructively interferes two Feynman paths, eliminating all signals generated by field interactions with four parallel transition dipoles, and the intense diagonal and bleach/stimulated emission peaks. At later delay times, θ
(t
) is adjusted to compensate for anisotropy caused by rotational diffusion. When implemented with TA spectroscopy or microscopy, the pump-probe spectrum is dominated by the cross-peak features. The local oscillator is also attenuated, which enhances the signal two times. This overlooked polarization scheme reduces spectral congestion by eliminating diagonal peaks in 2D spectra and enables TA spectroscopy to measure similar information given by cross-peaks in 2D spectroscopy.
We have recently developed a new and simple way of collecting 2D infrared and visible spectra that utilizes a pulse shaper and a partly collinear beam geometry. 2D IR and Vis spectroscopies are ...powerful tools for studying molecular structures and their dynamics. They can be used to correlate vibrational or electronic eigenstates, measure energy transfer rates, and quantify the dynamics of lineshapes, for instance, all with femtosecond time-resolution. As a result, they are finding use in systems that exhibit fast dynamics, such as sub-millisecond chemical and biological dynamics, and in hard-to-study environments, such as in membranes. While powerful, these techniques have been difficult to implement because they require a series of femtosecond pulses to be spatially and temporally overlapped with precise time-resolution and interferometric phase stability. However, many of the difficulties associated with implementing 2D spectroscopies are eliminated by using a pulse shaper and a simple beam geometry, which substantially lowers the technical barriers required for researchers to enter this exciting field while simultaneously providing many new capabilities. The aim of this paper is to provide an overview of the methods for collecting 2D spectra so that an outsider considering using 2D spectroscopy in their own research can judge which approach would be most suitable for their research aims. This paper focuses primarily on 2D IR spectroscopy, but also includes our recent work on adapting this technology to collecting 2D Vis spectra. We review work that has already been published as well as cover several topics that we have not reported previously, including phase cycling methods to remove background signals, eliminate unwanted scatter, and shift data collection into the rotating frame.
αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been ...linked with neurodegenerative diseases such as Alzheimer's, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin (Cryab-R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro, and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab-R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab-R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab-R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab-R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.
Abstract
Exciton-polaritons are hybrid states formed when molecular excitons are strongly coupled to photons trapped in an optical cavity. These systems exhibit many interesting, but not fully ...understood, phenomena. Here, we utilize ultrafast two-dimensional white-light spectroscopy to study donor-acceptor microcavities made from two different layers of semiconducting carbon nanotubes. We observe the delayed growth of a cross peak between the upper- and lower-polariton bands that is oftentimes obscured by Rabi contraction. We simulate the spectra and use Redfield theory to learn that energy cascades down a manifold of new electronic states created by intermolecular coupling and the two distinct bandgaps of the donor and acceptor. Energy most effectively enters the manifold when light-matter coupling is commensurate with the energy distribution of the manifold, contributing to long-range energy transfer. Our results broaden the understanding of energy transfer dynamics in exciton-polariton systems and provide evidence that long-range energy transfer benefits from moderately-coupled cavities.
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